ABSTRACT
Background: The nucleocapsid protein (N-protein) of SARS-CoV- 2 regulates transcription, replication and packaging of the viral genome. Potentially, each of the structural proteins can act as an antigen for the production of specific antibodies and T-cell stimulation. This study evaluates T-cellular immunity to N-protein after infection caused by the SARS-CoV- 2 for up to 9 months. Method(s): Patients who had COVID-19 in 2020 were divided by severity into 3 groups: mild (n = 41), moderate (n = 46), and severe (n = 29). Blood samples were taken 3 times: at 3, 6 and 9 months after infection. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained with Tag-it Violet vital dye. PBMCs were stimulated with PepTivator SARS-CoV- 2 ProtN (pool of peptides, covering the complete sequence of N-protein);unstimulated cells served as negative control. PBMCs were cultured in AIM-V media for 5-7 days. Cell colonies were detected by microscopy. Relative content (%) of proliferating live T cells (CD3+7AAD-) were evaluated by flow cytometry. Samples were counted positive for antigen-specific T cells (ASCs) to N-protein when both proliferation was >1% compared with negative control and microcolony were observed. Result(s): There were 58.5% positive samples with ASCs to the N-protein in mild, 60.9% in moderate and 48.3% in severe group in 3 months after recovery from COVID-19. There was no difference between groups at this point (p > 0.05, chi2). After 6 months results were: 26.7% positive samples in mild, 21.9% in moderate and 35.0 % in severe group. No differences between groups were observed after 6 months, but percentages of positive samples were less in mild and moderate groups when compared 3d and 6th months (p3-6 < 0.05, chi2). Drastic decreases of ASCs-positives in the mild group was observed after 9 month -only 4.2% (p6-9 < 0.05, chi2). Further decrease in moderate (25.0%) and severe (41.7%) groups was not observed after 9 months. Conclusion(s): T cell immunity to the N-protein of SARS-CoV- 2 was formed after mild, moderate and severe forms of COVID-19 with different effectiveness. The duration of immunity depends on the form of the disease and was stronger in severe group. By the 9th month, the number of positive samples in the group with mild COVID-19 was significantly lower when compared with the moderate and severe groups.
ABSTRACT
Introduction: Anti-SARS-CoV2 vaccination induces specific Tand B-cell responses in healthy subjects (HS). In MS patients treated with anti-CD20 drugs, the antibody response is reduced or absent, whereas specific T-cell responses are maintained. It is not known whether and how vaccination affects innate responses mediated by natural killer (NK) cells in HS and in MS patients treated with anti-CD20 drugs. Objective(s): To evaluate whether and how NK cells contribute to the immune response following anti-SARS-CoV2 vaccination in HS and in ocrelizumab-treated MS patients Aims: The aims of this work were: 1) to evaluate the effects of anti-SARS CoV2 vaccination on the phenotype of NK cells from HS and from ocrelizumab-treated MS patients and 2) to evaluate how peptides from the SARS-CoV2 spike protein affect NK cell responses before and after anti-SARS-CoV2 vaccination. Method(s): We enrolled 21 MS patients treated with ocrelizumab and 20 HS. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood and stored under liquid nitrogen. Thawed PBMCs were cultured overnight in presence/absence of SARS-CoV2 peptides or peptides from the cytomegalovirus (CMV), with/without activating cytokines. Phenotype of NK cells through a 13-marker flow cytometry panel and intracellular production of IFN-gamma were evaluated after culture. Result(s): Findings: 1) Vaccination increased the proportion of CD56dim NK cells in HS and MS patients. CD56posCD16neg NK cells, more abundant in MS patients before vaccination, decreased thereafter. Lower pre-vaccination activation capability of NK cells from MS patients compared to HS in response to stimulus with cytokines was reverted by vaccination. 2) Before vaccination, peptides from the SARS-CoV2 protein downregulated the production of IFN- gamma from NK cells of HS, but not ocrelizumabtreated MS patients, who had significantly lower baseline IFN-gamma NK cells 3) After vaccination, peptides from the SARS-CoV2 protein did not affect the production of IFN- gamma from NK cells of HS. Conclusion(s): The results of this work demonstrate anti-SARSCoV2 vaccination increases the proportion of effector CD56dim NK cells in HS and ocrelizumab-treated patients. Spike peptides inhibit the function of NK cells from HS before, but not after vaccination. Such phenomenon may contribute to the pathogenicity of SARS-CoV2 in unvaccinated subjects.
ABSTRACT
Introduction and Objectives Novel SARS-CoV-2 virus has been implicated in prompting a bold immune response that leads to severe Coronavirus disease 2019 (COVID-19). Recent studies have shown that SARSCoV-2-infected monocytes and macrophages are stimulated to produce an overabundance of pro-inflammatory cytokines and chemokines to generate a cytokine storm. Cytokines in excess can contribute to local tissue inflammation and the pathogenesis of COVID-19. However, the mechanism by which SARS-CoV-2 signal macrophage-derived inflammatory response remains unclear. In the present study, we used RAW 264.7 cells, a wellcharacterized macrophage model, to study the in vitro effects of SARS-CoV-2 on reactive oxygen species (ROS) production and its potential role in the signal transduction of cytokine production. Methods The effect of SARS-CoV-2 on ROS and cytokine generation in macrophages was assessed by treating RAW 264.7 cells with SARS-CoV-2 heat inactivated virus (0-20 million viral particles) or recombinant proteins for 24 hours. 2',7'-Dichlorodihydrofluorescein (2',7'-DCF) fluorescence analysis was utilized to quantify ROS generation within the RAW 264.7 macrophage cell line. Cell culture medium was sampled to quantify the levels of tumor necrosis factor (TNF) using enzyme-linked immunosorbent assay (ELISA). To assess the effects of SARS-CoV-2 on mitochondrial function, cells were treated with SARS-CoV-2 heat inactivated virus (0-20 million viral particles) for 24 hrs. Mitochondria-derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator. Results Treatment of RAW 264.7 cells with inactivated SARS-CoV-2 viral particles or recombinant proteins stimulated ROS production. Mitochondria-derived superoxide and hydrogen peroxide production were increased in response to inactivated SARS-CoV-2 viral particles and recombinant protein exposure. The increased ROS generation is linked to macrophage activation induced by SARS-CoV-2 exposures. Along with the ROS generation, increased TNF production was observed. Conclusions The results of this study suggest that both SARS-CoV-2 viral proteins and heat-inactivated viral particle exposures cause significant generation of ROS and cytokines by RAW 264.7 cells. ROS generation and the subsequent cytokine release apparently play a significant role in the pathogenesis associated with the SARS-CoV-2 viral infection. The imbalanced cellular defense system against oxidative stress commonly associated with aging could explain the increased occurrence of more severe SARS-CoV-2 illness in seniors and in patients with underlying health conditions. Based on the results from this study, we propose that antioxidants such as N-acetyl-L-cysteine, resveratrol, or Vitamin E in combination with antiinflammatory drug could be used to control excess ROS and cytokines in patients with severe COVID-19.
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Objectives: (1) Present a case of visual decompensation related to a Rathke's cleft cyst (RCC) in setting of acute COVID-19 infection. (2) Review the literature on the presentation and management of RCCs. Study Design: Case report and review of the literature. Results: We report a case of a 32-year-old female diagnosed with a sellar mass who was transferred from an outside hospital for acute visual field decompensation. Upon arrival, the patient reported a decline in vision over the past 3 days, photophobia, and diplopia with physical exam confirming these findings. Of note, patient was unvaccinated and laboratory testing revealed that she was COVID-19 positive with mild symptoms of congestion. Imaging revealed a large intrasellar and suprasellar T1 hyperintense mass with displacement of the optic chiasm superiorly ([Fig. 1]). An endoscopic transsphenoidal approach was performed and the cyst was marsupialized into the sphenoid sinus decompressing the optic chiasm. Cultures demonstrated many white blood cells, but no organisms. Pathology confirmed a diagnosis of RCC. There was no intraoperative cerebrospinal fluid leak noted. To prevent reaccumulation of cyst contents, a free mucosal graft harvested from the posterior septectomy and was used to line the sellar defect to facilitate marsupialization into the sphenoid sinus. Postoperatively, physical exam revealed normalization of her vision and extraocular movements, and the patient subjectively confirmed these findings. She was discharged home on postoperative day 3 without complications. Review of the literature demonstrates that visual field deficits are a common presenting finding in RCC patients and acute inflammation of RCCs can present similarly to pituitary apoplexy. Transsphenoidal approaches are safe and effective in surgical decompression of RCCs. Lining the exposed cyst cavity with a free mucosal graft or nasoseptal flap may facilitate promotion of an epithelialized track to prevent cyst reaccumulation. Conclusion: A case of acute visual decompensation related to a RCC in setting of acute COVID-19 infection is presented. Inflammation of the RCC, presumably related to COVID-19 infection, contributed to the acute presentation. Endoscopic transsphenoidal approaches are effective at decompression of RCCs and free mucosal grafts or nasoseptal flaps may promote epithelialization of a tract to decrease recurrences.
ABSTRACT
Introduction: Laboratory parameters are crucial in diagnosis and prognosis of Coronavirus Disease 2019 (COVID-19). It would be of interest to explore morphological changes in infected White Blood Cells (WBCs). A detailed examination of peripheral smears may shed light on pathophysiology of infected cell lines and differentiate them from those in established viral infections like dengue and infectious mononucleosis. Aim: To study morphological changes of WBCs in peripheral smears of severe and non severe cases of COVID-19 patients. Materials and Methods: This cross-sectional study was conducted at a tertiary care centre, Dr. Chandramma Dayananda Sagar Institute of Medical Education and Research, Ramanagara, Karnataka, India, from April 2021 to August 2021 on 120 peripheral smears of adult COVID-19 positive cases. Abnormal morphological features were graded by counting 100 cells in each of neutrophils, lymphocytes and monocyte lineage. Changes were compared and analysed between severe and non severe groups using Statistical Package for Social Sciences (SPSS) software version 26.0. A p-value <0.05 was considered as significant. Results: The study included total of 120 cases (59 severe and 61 non severe) with a mean age of 47 years. Male to female ratio in severe and non severe categories were 1:1.2 and 1:0.6, respectively. Severe category patients (n=59) were associated with statistically significant leucocytosis (p-value=0.04), absolute neutrophilia (p-value=0.03) and higher grades of morphological changes- abnormal nuclear morphology (p-value=0.002) and Pseudo-Pelger-Huët anomaly in neutrophils (p-value=0.029), plasmacytoid lymphocytes (p-value=0.03), cytoplasmic granularity and atypical lymphocytes (p-value=0.04). Monocytes showed large coalescent vacuoles and cytoplasmic granules (p-value=0.03). Though present in non severe category (n=61), they were proportionately of lesser grades. Conclusion: Viral cytopathic effects in WBC cell lines on peripheral smear had significant clinical implications on disease severity, undermining need for a comprehensive study of viral induced morphological changes in hospitalised COVID-19 patients.